The proposed work is aimed at elucidating the role that immune reactive cells, monocytes and lymphocytes, play in the fibrin deposition that is characteristic of several inflammatory lesions. Their ability to participate in the molecular events leading to the generation of the clotting enzyme, thrombin, will be assessed. Thrombin is produced by the enzymatic complex, Prothrombinase - the enzyme, Factor Xa, the cofactor Factor Va, Ca++ and a "surface membrane" for proper assembly of the protein components. The ability of monocytes and lymphocytes to provide a functional catalytic "surface membrane" will be determined by: 1) equilibrium binding techniques to measure the interaction of radiolabeled Factor V, Factor Va and Factor Xa with the surface of these cells; and 2) a kinetic assessment of the ability of these cells to facilitate the formation of a functional Prothrombinase complex as measured by thrombin formation. Thrombin formation will be monitored using dansyl-arginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) which, upon binding to thrombin, yields a change in fluorescence intensity providing a continuous monitor of thrombin concentrations. The ability of these cells to provide Factor V (the cofactor required for Prothrombinase complex assembly) will also be determined. Mixed mononuclear cell (MNC) cultures stimulated with various agents show increased procoagulant activity (PCA) which has been attributed to monocyte production of tissue factor. Equilibrium binding techniques and kinetic experiments will be used to determine if this increased PCA is paralleled by an increase in monocyte and lymphocyte Prothrombinase activity. Freshly-isolated monocytes, stimulated with conditioned medium obtained from MNC cultures previously stimulated with phytohemagglutinin, Concanavalin-A, lipopoly-saccharide, and various lipoprotein classes, will be assessed for their ability to form functional Prothrombinase complexes. Adherent monocyte cultures will be stimulated in an analogous manner to determine if the onset of the monocyte differentiation process is a prerequisite for the appearance of the observed PCA. Lymphocytes isolated from the stimulated MNC cultures will also be assessed for their ability to activate prothrombin. It is anticipated that these studies will provide insight into cellular and molecular events which may begin to explain the relationship between coagulation and immune reactive cell accumulation in the pathogenesis of inflammatory lesions.